An immunoinformatics study reveals a new BoLA-DR-restricted CD4+ T cell epitopes on the Gag protein of bovine leukemia virus

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis (EBL), which has been reported worldwide. The expression of viral structural proteins: surface glycoprotein (gp51) and three core proteins - p15 (matrix), p24 (capsid), and p12 (nucleocapsid) induce a strong humoral and cellular immune response at first step of infection. CD4+ T-cell activation is generally induced by bovine leukocyte antigen (BoLA) region– positive antigen-presenting cells (APC) after processing of an exogenous viral antigen. Limited data are available on the BLV epitopes from the core proteins recognized by CD4+ T-cells. Thus, immunoinformatic analysis of Gag sequences obtained from 125 BLV isolates from Poland, Canada, Pakistan, Kazakhstan, Moldova and United States was performed to identify the presence of BoLA-DRB3 restricted CD4+ T-cell epitopes. The 379 15-mer overlapping peptides spanning the entire Gag sequence were run in BoLA-DRB3 allele-binding regions using a BoLA-DRB- peptide binding affinity prediction algorithm. The analysis identified 22 CD4+ T-cell peptide epitopes of variable length ranging from 17 to 22 amino acids. The predicted epitopes interacted with 73 different BoLA-DRB3 alleles found in BLV-infected cattle. Importantly, two epitopes were found to be linked with high proviral load in PBMC. A majority of dominant and subdominant epitopes showed high conservation across different viral strains, and therefore could be attractive targets for vaccine development.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leucosis (EBL), a chronic, lymphoproliferative disease associated with persistent lymphocytosis and B-cell lymphomas 1 .BLV, together with human T-cell leukemia viruses type 1 and 2 (HTLV-1, HTLV-2), belong to the genus Deltaretrovirus of the family Retroviridae.BLV infection has a worldwide distribution and causes substantial economic losses in the livestock industry 2,3 , infection with this virus result in a negative effect on dairy production and cow longevity, which is very likely based on the resulting impaired immune function following infection [3][4][5] .
The complex BLV genome encodes structural genes (env, gag, pol/pro) and nonstructural, regulatory genes (tax, rex).The env gene gives rise to two glycoproteins: extracellular surface subunit (SU, gp51) implicated in receptor recognition and virion attachment, and transmembrane subunit (TM, gp30) responsible for anchoring the SU-TM complex into lipid bilayers (reviewed in 6 ).
The gag gene encodes the internal structural polyprotein-Gag (group-specific antigen), responsible for initiating the process of virion budding from the infected cell and RNA packaging in the viral particle formation process 7,8 .During viral maturation, the precursor Gag is processed into three separate proteins: matrix (MA, p15), capsid (CA, p24) and nucleocapsid (NC, p12), which undergo substantial conformational rearrangements-and

Analysis of amino acid sequence variability of the Gag protein
The Shannon entropy (Hx) plot exhibited 49 peaks with values ranging from 0.05 to 1.13 (Supplementary Fig. S1, Supplementary Table S1).Considering the three Gag domains, the highest total entropy (6.33) occurred in the matrix (MA).For the capsid (CA) and nucleocapsid (NC) domains, the total entropy was 4.09 and 1.38, respectively.A detailed multiple sequence alignment analysis on 395 amino acid sites indicated 345 as conserved and 49 with non-synonymous single nucleotide polymorphisms (nsSNPs).A substantial number of nsSNPs found in the MA and CA domains suggested the possibility of positive selection on variable sites of the protein (Supplementary Fig. S2).The dN/dS ratios were drawn over the midpoint window position (window length 9, step size 3) from the whole coding region.The following regions with putative positive selection sites were identified: 133-149 nt, 175-195 nt, 199-216 nt, 250-279 nt, 316-342 nt in MA, 829-843 nt, 952-972 nt in CA and 1087-1107 nt for NC domains, respectively.Thirteen codons located in these regions had dN/dS ratios > 1 that identified them as major sites for the occurrence of positive selection.These were codons 48, 61, 63, 69, 87, 88, 108, 109, 112 in MA domain; 278, 318, 323 in CA; and 365 in NC (Supplementary Table S2 and Supplementary Fig. S1).

Determination of epitope peptides in the Gag protein consensus sequence based on BoLA-DRB3
In order to search for common epitope peptides on Gag protein, we determined the consensus sequence from 125 sequences described in this study.To detect putative binding sites for BoLA-DRB3, we used the 379 15-mer overlapping peptides that spanned the entire Gag consensus sequence in 73 BoLA-DRB3 allele binding regions in NetBoLAIIpan (pan-specific predictor for BoLA-DRB3 Ag presentation).Analysis revealed 22 putative regions within Gag proteins with high binding affinity to BoLA-DRB3 alleles.The binding affinities of the epitopes and complete calculations are presented in Supplementary Table S3.

Relation between the incidence of BoLA-DRB3 alleles and number of CD4+ T-cell epitopes
Predictions of BoLA-DRB3 peptide's binding affinity were performed for 73 alleles determined in BLV-infected cattle tested in this study.The results shown in Supplementary Table S4 indicate that the examined alleles had significant binding specificity to the epitope peptides.BoLA-DRB3 molecules were found to interact with binding core sequences of 3  S4).The combined incidence of these alleles was 72 out of 237 possible pairs (30.4%).Conversely, the first ten alleles distinguishable by binding affinity for the highest number of Gag epitopes were *006:01 (eleven epitopes); *03:01:01 (ten epitopes); *24:32, *24:33, *57:02, *005:08 and *80:01 (nine epitopes); and *09:01, *02:01 and *24:03 (eight epitopes) (Supplementary Table S4).In total, the incidence of these alleles was 21 out of 237 (8.9%).These results indicate that it needs to be determined if there is a relation with the number of binding sites versus susceptibility for BLV or progression to clinical disease.
Next, the numbers of epitopes were analyzed with respect to proviral copy numbers in BLV-infected cattle.Figure 3 depicts results of such analysis and demonstrates that the number of CD4+ T-cell epitopes on Gag protein identified for different BoLA-DRB3 alleles is not significantly correlated with BLV proviral load (R 2 = 0.0231, P value = 0.0793, n = 113).
As a result of the analysis, none of Group A alleles had affinity for epitopes 1A and 2 (Fig. 4), in contrast to group B alleles' significant affinity for epitopes 1A and 2. For other epitopes, allele binding from the two groups did not differ and no other patterns were observed between the two groups as far as binding affinity of alleles to these epitopes (Supplementary Fig. S4).Subsequently, the affinity or lack of affinity for epitopes 1A and 2 was determined for the remaining 63 alleles ( 63 01.Thus, one might infer that Group A alleles' lack of affinity for epitopes 1A and 2 is related to the number of BLV proviral copies in BLV-infected cattle.Therefore, we conducted a BLV copy number comparison of cattle carrying at least one Group A allele and those with only Group B alleles.Statistical analysis using the student t-test showed that cattle carrying one or both Group A alleles (with no affinity for 1A and 2 epitopes) had a significantly increased number of BLV proviral copies per 1000 cells, as opposed to Group B alleles (t-value = 2.06255, P value = 0.040269) (Fig. 5 and Supplementary Table S5).

Changes in amino acid sequence of Gag vary BoLA-DRB3-peptide binding affinity
The 13 codons, which were identified as the major sites with a process of positive selection were evaluated for epitope affiliation.Out of these 13, 11 codons (84.6%) were located in the following epitopes: codon 48 in epitopes 4A and 6; codons 61 and 63 in epitope 11A; codons 69, 87 and 88 in epitope 5; codons 108, 109 and 112 in epitope    S6).Detailed descriptions of the changes on epitope peptides, and the BoLA-DRB3 epitope binding level are shown in Table 3 and Fig. 6.

Analysis of sequence conservation in the binding core of the CD4 + T-cell epitopes
The binding core is the anchoring region of the epitope and is defined as the central nine amino acid (aa) sequence of the predicted 15-mer epitope that is flanked by three aa residues on the N-and C-terminal ends.Therefore, the conservation of the 22 identified putative CD4+ T-cell epitopes was evaluated based on the number of changed amino acid residues in their binding-core sequences (Table 4).The most conserved epitope cores were present in 1B (CA), 4B (CA), 4C (CA), 7 (CA), 8 (NC), 12A (CA), 13A (CA), 14 (CA) and 15A (MA) epitopes, with complete sequence conservation in 125 sequences (100%).Core sequences of epitopes 15B (CA), 2 (CA), 10 (CA) and 12B (CA) showed the second highest level of conservation, in the range of 96.8% (in 121 of 125 sequences) to 99.2% (in 124 of 125 sequences), respectively.Importantly, the mutations found in these core sequences were predicted to no affect the epitope binding affinity to the BoLA-DRB3 molecules (Table 3).Epitopes 1A (CA/NC), 5 (MA), 9 (CA), 3 (CA) and 13B (CA) showed the conservation in the range of 87.2% (in 109 of 125 sequences) to 92.8% (in 116 of 125 sequences), respectively, and contained the mutations I323V, V254I, A78T, E82D, V76I, A193T, and A25T, which may affect epitope binding affinity to certain BoLA-DRB3 molecules.The variable epitopes were 11A (in 59 of 125 MA sequences, 47.2%), 4A and 6 (in 94 of 125 MA sequences, 75.2%) and 11B (in 98 of 125 MA/CA) sequences, 78.4%), containing amino acid changes H48R/Y, G61S, A63V/T, L109M, I112V and V108I, which were predicted to affect binding to BoLA-DRB3.

Discussion
The utilisation of bioinformatics to identify T-cell responses to retroviral infections has increased over the last few years [34][35][36][37][38] .The in silico prediction methods NetMHCIIpan and NetBoLAIIpan developed to predict HLA and BoLA class II restricted peptide binding, respectively, have proven to be among the best methods currently available 35,39,40 .Here, we used NetBoLAIIpan prediction method to determine BoLA-DRB3-restricted BLV peptides on p15, p24 and p12 of the Gag polyprotein, with broad BoLA allelic coverage.Of all tested epitope candidates, 11 top-scoring epitopes (1A, 1B, 2, 3, 4A, 4B, 4C, 5 -8) were selected to identify broadly reactive BLV-specific CD4+ T-cell responses, by up to 60% of the analysed BoLA-DRB3 alleles.Another 11 subdominant www.nature.com/scientificreports/epitopes (9, 10, 11A, 11B, 12A, 12B, 13A, 13B, 14, 15A, 15B) were predicted to be more restricted to one or more of the donor's BoLA class II alleles, up to 17.8% of all examined BoLA-DRB3 alleles.These identified epitopes often overlapped, thus creating long regions within the Gag protein that had BoLA-DRB3 binding affinity.In MA, dominant epitopes occupied 40%, in CA 57% and in NC 47% of the protein sequences.Importantly, the Gag peptide-binding motifs were detected for all bovine BoLA-DRB3 proteins (n = 73) recognized for the 125 cows used in the current studies.Each of the alleles had at least three of the identified target epitopes.The presented data demonstrates a high promiscuity of Gag protein to BoLA-DRB3.BoLA-DRB3-restricted CD4+ T-Cell epitopes 1A and 1B were considered the most promiscuous binders as they contained binding cores of epitopes predicted for 44 different BoLA-DRB3 types.They were located within a conserved part of the p24 region, C-terminal domain, which is required for capsid dimerization, Gag oligomerization and viral formation [41][42][43]  ).We noted that the previously defined epitopes were nested within newly identified epitopes.Therefore, we strongly suggest that these regions may be alternatively extended by the amino acids contained in the epitopes defined by BoLA-DRB3 II peptide-binding prediction method.Interestingly, in contrast to the second epitope (residues 141-165) region, epitopes 1B, 3 and 13B cover the full length of the major homology region (MHR) 244-IVQGPAESYVEFVNRLQISL-263, which was found to be essential for the stability and folding of the monomer, and hence for viral assembly, maturation and infectivity.This region is conserved throughout the whole retrovirus group and thus offers a novel and stable target for viral vaccines.
We observed that a higher number of Gag protein epitopes recognized by certain BoLA-DRB3 alleles accompanied the alleles associated with BLV resistance in cattle.Interestingly, relatively few of them were observed in the population of BLV-infected animals.Likewise, fewer epitopes recognized by particular BoLA-DRB3 alleles were associated with the BLV susceptible alleles.Noteworthy, the percentage of these alleles in the examined population of virus-infected cows was relatively high.Indeed, the affinity for the interaction of certain BoLA-DRB3 alleles with a longer region of the Gag protein (where epitopes overlap) or more Gag regions may elicit a stronger cellular response.Thus, our results confirm the hypothesis that disease-susceptible cattle may have fewer epitopes than resistant cattle, resulting in weaker immune responses.Moreover, these results indicate a significant role of bovine MHC II polymorphisms in the mapping of BLV epitopes recognized by CD4+ T-cells on viral proteins.
Bai and coworkers studied gp51, gp30 and Tax protein epitopes related to the BoLA-DRB3 genotype and found that fewer CD4+ T-cell epitopes were observed in susceptible cattle than in resistant cattle 19 .Takeshima and colleagues suggested that the BoLA-DRB3 gene may regulate both antigen epitope recognition and the magnitude of the antigen-specific T-cell response that is processed after exposure to infection 23 .Accordingly, our studies confirm that BLV antigens are restricted according to BoLA-DRB3, and that genotyping of cattle is important for determining antigenic epitopes recognized by the bovine immune system.
In this work, we also analyzed whether the number of BoLA-DRB3-restricted epitopes in Gag protein is related to the number of BLV proviral copies in PBMCs in the analyzed DNA samples; however, we did not find a significant correlation.Additionally, Bai and colleagues observed that the number of CD4+ T-cell epitopes was positively related to proviral load, which depended on the BoLA class II genotype 19 .This discrepancy may be due to the fact that the current study used a 25-fold larger group of cattle for the analysis.It is well known that the BLV proviral load varies greatly as it is the result of many different factors such us the time of exposure to the virus, biochemical and hematological factors of the cow, or the age of the cow, to name a few, which can generate erroneous results when experiments are conducted on a small number of animals [44][45][46]  Decreased binding affinity for 1A epitope Table 3. Changes in the amino acid sequence of the Gag peptides that alter the binding affinity of the BoLA-DRB3 alleles.To specify the level of binding affinity prediction for peptide-BoLA-DRB3 complexes the following colors were used: weaken binding affinity is marked in green; higher binding affinity is marked in orange; new binding affinity is marked in red; amino acid changes that remove epitope binding affinity are marked in grey.*-peptide position out of 379 isolated peptides along Gag; ∂ NB-Non-binders (%Rank) > 5%; ∞ SB-strong binding peptides (%Rank) < 1.0%; ∆ WB-Weak binding peptides (%Rank) < 5%.
lower viremia in both adults and children [47][48][49] .Ranasinghe and coworkers demonstrated an inverse correlation between viral load and the number of Gag peptides targeted by CD4+ T-cells 50 .Buggert and coworkers confirmed this finding, suggesting that broadly reactive Gag-specific CD4+ T-cell responses could have an impact on HIV disease progression 51 .However, whether the frequent targeting of Gag peptides is the cause or the consequence of the reduced viremia remains to be clarified.Nevertheless, an association polymorphisms of the BoLA-DRB3 gene with BLV PVL is described in the literature 23,31,45,52,53 .Published data indicates some BoLA-DRB3 alleles such as *15:01, *12:01 and *16:01 are associated with high PVL in BLV-infected cattle but BoLA-DRB3 alleles like *09:02, *02:01 and *14:01:01 are associated with low PVL [29][30][31][32] .Of the 22 epitopes, we found two epitopes-1A (317-KIKQPAILVHTPGPKMPGPR-336) and 2 (293-ILQGRGLVAAPVGQKLQACA-312) that were significantly related to cattle resistant to developing www.nature.com/scientificreports/high BLV proviral load.These epitopes were located in CTD-CA and between CA/NC proteins, respectively, highly conserved regions for retroviruses (Supplementary Fig. S6).In addition, these epitopes were broadly recognized for most of the BoLA-DRB3 alleles (70%).Interestingly, epitopes 1A and 2 were not recognizable by the types of BoLA-DRB3 alleles, which were previously reported in the literature as being associated with the development of subclinical infection and high BLV PVL [29][30][31]54,55 . Some f these have never been investigated for PVL dependence therefore, additional functional studies are required to further confirm these findings.Nevertheless, in the case of HIV, there are certain epitopes that determine resistance to infection 56 .Our results suggest that the 1A and 2 epitopes may have a key and powerful effect in inducing a strong cellular response and fighting BLV within the host.It is noteworthy that epitopes 1A and 2 were the strongest epitopes to which the most alleles bound.Therefore, they seem to be an indispensable element that would be instructive in the design of synthetic peptide vaccine.Antigenic variation within T-cell epitopes has been demonstrated for HIV-1, and this 'antigenic escape' may be responsible for viral persistence.Generally, although external proteins are highly immunogenic, antigenic shift limits their capacity to provide cross-protective immunity to novel viral strains.In contrast, the internal proteins are more conserved and may better mediate cross-protective T-cell responses 57,58 .
BLV exhibits less genetic variation among strains as compared with most other retroviruses, and the genomes of viruses isolated from multiple countries around the world share approximately between 94.5 and 99.5% of their nucleotide sequences.However, variation within the sequences encoding the Gag protein is poorly characterized.In our study the pairwise identity for 125 gag nucleotide sequences was 97.3%.Despite the internal proteins MA, CA and NC that exhibit higher levels of conservation relative to SU (gp51), sequence variation was still present, in which most sequence variation can be attributed to a single mutation.Based on the resulting proviral mutation profile, we revealed that the mutations are driven by immune selection pressure, suggesting mechanisms of positive selection and mutation hotspots.
Diversified positions were preferentially located within bovine CD4+ T-cell epitopes.Of 13 hotspots, 7 were located in the 9-mer core epitopes and had predicted a significant effect on the binding affinity of BoLA-DRB3 molecules.This is consistent with what is known about the peptide-binding core of epitopes that primarily interact with the BoLA-DR antigen binding groove.The peptide-BoLA-DR binding affinity is primarily determined by the amino acid sequence of the peptide binding core 59 .However, it has been shown that peptide flanking regions (PFRs) on either side of the binding core affect peptide-BoLA-DR binding and thereby ultimately also influence the peptide immunogenicity 40 .Indeed, 4 hotspots were located in PFRs and have input on binding affinity.These mutations may upend the presentation of virus-derived peptides via BoLA-DR.Based on the obtained results, certain mutations reduced while other mutations increased the affinity to bovine MHCII.Additionally, some mutations exhibited a neutral affinity.On the basis of our analysis of mutations, we selected 12 mutant peptides with predicted decreased BoLA-DR-binding strength 19 and mutant peptides with predicted increased Table 4.The degree of evolutionary conservation of an amino acids in a core sequences of predicted 22 potential CD4 + T-cell epitopes on Gag protein interacting with different BoLA class II alleles.The table shows the amino acids in the core sequences and peptide flanking regions, which affect peptide-MHC binding and, thereby ultimately also influence the peptide immunogenicity.BoLA-DR-binding strength for further biophysical and functional analyses.Our study provides evidence that single nonsynonymous mutations in BLV can subvert the immune response to CD4+ T-cell epitopes.
Our hypothesis was that the substitution of single amino acids in CD4+ T-cell epitope may influence the BoLA-DRB3 binding affinity and that nsSNP might be associated with variations in individual immune responses to antigens and susceptibility or resistance to disease.There are therefore many factors that make it difficult to predict peptide binding affinities to BoLA-DRB3 molecules, including the polymorphic sites of Gag epitopes.However, without functional analysis, the impact of single anchor residue substitutions on the response of CD4+ T-cells is still unclear.This study does not allow direct conclusions to be drawn concerning potential selection pressures, which shape the mutational landscape of CD4+ T-cell epitopes.This would invariably involve accounting for the BoLA-DRB3 genotype of all individuals from whom BLV genomes were sequenced.Moreover, how T-cell escape mutations within BLV are maintained during virus transmission between individuals with differing BoLA types and how viruses carrying epitope mutations affect disease severity requires further investigation.
Many CD4+ T-cell epitopes for BLV have been described in this study.The CD4+ T-cell response against BLV was associated with broad epitope recognition of, on average, 6 CD4+ T-cell epitopes per antigen per BoLA-DRB3 allele, which raises the question whether and how mutations in single epitopes affect virus control 60 .This may be of particular importance for BLV subunit vaccines to induce responses against an unlimited number of CD4 epitopes.These results highlight the capacity of BLV to evade cellular immune responses through sporadically emerging mutations in BoLA-BRB3 epitopes.
Taking into account the very conservative and wide range of identified epitopes and, on the other hand, the lack of progress in obtaining an effective vaccine, the new discovery has a high chance of success.The new vaccine could be an important element in protecting herds against BLV infections, especially in dairy cattle, where this category of cattle is especially susceptible to BLV infection 61 .Moreover, preventive vaccinations based on selected peptide immunogens could become an integral part of BLV eradication programs 62,63 .Finally, the importance of immunopeptidomics should be emphasized in subsequent studies taking into account other exotic and local cattle breeds as well as the circulation of endemic BLV variants.

Conclusions
In the present study, BLV Gag protein was characterized by immunoinformatic techniques to identify potential T-cell epitopes.Twenty-two BoLA-DRB3 class II epitopes were available across the entire BLV Gag polyprotein, however the p24 protein was identified as the main target for recognition by antigen-specific CD4+ T-lymphocytes.The thirteen broadly conserved BoLA-DRB3-restricted CD4+ T-cell epitopes shared between BLV isolates from different countries and 9 epitopes with changes in the binding core were identified.Among them two promiscuous conserved pBoLA-(gag)peptides, 1A and 2, related to hosts that mounted a successful host-pathogen immune response (animals with low proviral load) were discovered.We believe the newly-identified pBoLA-(gag) peptides, together with additional peptides that have been shown within gp51, gp30 and Tax proteins, will be important for inclusion in a multivalent antigen peptide vaccine for BLV that can provide protection against BLV infection caused by geographically distant viral strains in cattle that express different BoLA class II DRB3 haplotypes.

Ethics declaration
The study was approved by the Veterinary Sciences Animal Care Committee No. AC21-0210, Canada; the Institutional Animal Care and Use Committee No. PROTO202000096 from 4/13/2020 to 4/14/2023, Michigan State University, United States; the Ethics Review Board, COMSATS Institute of Information Technology, Islamabad, Pakistan, no.CIIT/Bio/ERB/17/26 and the Bioethics Commission No. 06-18 on 30 January 2018, Almaty, Kazakhstan.Blood samples from Polish and Moldovan cattle, naturally infected with BLV, were selected from collections at local diagnostic laboratories as part of the Enzootic bovine leukosis (EBL) monitoring program between 2012 and 2018 and sent to the National Veterinary Research Institute (NVRI) in Pulawy for confirmation study.The approval for collection of these samples from ethics committee was not required according to Polish regulation ("Act on the Protection of Animals Used for Scientific or Educational Purposes", Journal of Laws of 2015).

Sample collection and preparation
A total of 125 DNA samples obtained from blood of naturally BLV-infected cattle from Canada, United States, Poland, Moldova, Pakistan and Kazakhstan were used for this study.Seventy-six of them were archival DNA samples obtained between 2013 and 2018 as described in our previous studies on samples from Poland (n = 22) 64,65 , Moldova (n = 14) 66 , Pakistan (n = 20) 67 and Kazakhstan (n = 21) 68 .Between 2020 and 2021 48 peripheral blood and serum samples from naturally BLV-infected cattle were obtained from three dairy farms of Alberta, Canada and two dairy farms of Michigan, US (see Table 1).All cattle were positive for anti-BLV antibodies, as determined by commercially available ELISA kit (IDEXX Leukosis Serum X2 Ab Test, IDEXX).Genomic DNA were isolated using a Quick DNA Miniprep Plus kit (Zymo Research) and a DNeasy Blood & Tissue Kit (Qiagen) for Canadian (n = 24) and US (n = 24) whole blood samples, respectively, following the manufacturer's protocol.

Figure 1 .
Figure 1.Distribution of the BoLA-DRB3-restricted CD4+ T-cell epitopes along the Gag polyprotein.The labeled blue bars in the upper part of figure refer to the identified 22 epitopes 1A-15B.The figure shows the localization of the epitopes for the most commonly detected BoLA-DRB3 (on the left side of the figure).The distribution of the epitopes for the all analysed alleles is shown in Supplementary Fig. S3.◀

Figure 4 .
Figure 4. Association between 73 BoLA-DRB3 alleles and two BoLA-DRB3-restricted CD4 + T cell epitopes (1A and 2).Log2 Rank predicted binding score for Gag peptides observed for the BoLA-DRB3 alleles distinguished in the two groups: Group A (n = 22 alleles) marked in red line on the graph; Group B (n = 51 alleles) marked in grey line.

Figure 5 .
Figure 5.Comparison of BLV copy number between cattle carrying BoLA-DRB3 alleles with no affinity to the Gag protein CD4+ T-cells 1A and 2 epitopes on and alleles with strong affinity to the epitopes using the student t-test for 2 independent means.

Figure 6 .
Figure 6.Gag protein sequence alignment for selected BLV isolates, containing amino acid changes within CD4+ T cell epitopes that change the degree of binding affinity of BoLA-DRB3.The names of the isolates and their corresponding BoLA-DRB3 are listed on the left side of the alignment.Amino acid changes, which generate new BoLA-DRB3 binding affinity site are marked with red arrows; amino acid changes that impair BoLA-DRB3 binding affinity sites are marked with green arrows; changes that enhance BoLA-DRB3 affinity are marked with orange arrows, the changes that generate lack of peptides interactions with BoLA-DRB3 are marked with grey arrows.BoLA-DRB3-restricted CD4 + T-cell epitopes along the Gag polyprotein are labeled in the upper part of the figure as blue bars.

Table 1 .
Characterization of BLV-infected cattle in this study.

Table 2 .
CD4+ T cell epitope peptides found in BLV Gag sequences.In bold type are marked the peptide sequences showing the most frequent affinity for BoLA-DRB3 alleles in particular Gag regions.